Xanthone analogs for treating infectious diseases and complexation of heme and porphyrins

ABSTRACT

Therapeutic compounds and compositions for the treatment of infectious diseases are disclosed. The compounds are xanthones and xanthone derivatives, such as 3,5-bis-ε-(N,N-diethylamino)amyloxyxanthone. The described compositions include such compounds and a pharmaceutical carrier. These compositions also can include additional materials conventionally used to form therapeutic compositions. 3,5-bis-ε-(N,N-diethylamino)amyloxyxanthone has an IC 50  for Plasmodium falciparum of about 0.15 μM, and an IC 50  for Leishmania mexicana of &lt;&lt;0.5 μM. These compositions are additionally useful for forming soluble complexes with heme and porphyrins.

ACKNOWLEDGMENT OF U.S. GOVERNMENT SUPPORT

[0001] This invention was made with partial support from the UnitedStates Government to Drs. Michael K. Riscoe and David J. Hinrichsthrough the Veterans Affairs Merit Review System. The U.S. Governmentmay have certain rights to this invention.

BACKGROUND

[0002] Protozoan parasites cause diseases such as malaria,trypanosomiasis, Chagas' disease, leishmaniasis, giardiasis, andamoebiasis. These and other parasitic diseases historically haveoccurred in tropical and sub-tropical areas where they cause widespreadsuffering of human populations. Although they receive little attentionin the Western world, protozoan diseases affect more people worldwidethan diseases brought on by any other biological cause (Heyneman, 1988).

[0003] Today, malaria remains the most destructive single infectiousdisease in the developing world. It is responsible for more human death,energy loss, more debilitation, more loss of work capacity, and moreeconomic damage than any other human ailment facing the world today(Heyneman, 1988). The World Health Organization estimates that 1 to 2million deaths are caused by malaria each year in Africa alone; most ofthese are children under the age of five (World Health Organization,1991). In addition, over 300 million people worldwide are believed to bechronically infected, and each year nearly one third of theseindividuals will suffer acute manifestations of the disease.

[0004] Today, the pathologic capacity of protozoa is being increasinglydemonstrated in the Western world among AIDS (Acquired ImmunodeficiencySyndrome) victims. AIDS depletes the immune system of affectedindividuals. This allows opportunistic agents to infect AIDS patients,which agents otherwise would be defeated by an active immune system.Several protozoa have proved especially opportunistically infectious inAIDS patients, including Cryptosporidium parvum, Entamoeba histolytica,Giardia lamblia, Pneumocystis carinii (which may be a fungal orprotozoal pathogen), and Toxoplasmosis gondii.

[0005] Despite the prevalence and significance of protozoan infections,therapy for these diseases is generally poor or in need of improvement.Many chemotherapeutic agents used to treat protozoan infections arenon-specific cytotoxins that are highly toxic and cause severe sideeffects in patients. However, these drugs are used because there are nobetter alternatives. For example, giardiasis and amoebiasis are treatedusing metronidazole (a nitroimidazole), but the mutagenic potential ofthis drug (Campbell, 1986) and its adverse interaction with alcohol areproblematic. For trypanosomiasis and leishmaniasis standard therapies(suramin, melarsoprol, and pentavalent antimonials) are dangerouslytoxic, occasionally fatal, and often ineffective (Mebrahtu, 1989; Groglet al., 1992). Other drugs are becoming ineffective due to emergingresistance. In the case of malaria, effective therapy previously hasbeen provided by chloroquine but its efficacy is now threatened by therapid emergence of drug resistant strains of Plasmodium falciparum, thecausative agent for the most severe, often fatal, form of the disease(Cowman, 1990). Other protozoal infections such as cryptosporidiosis orChagas' disease have no proven curative agent.

[0006] New therapeutic agents have been developed to treat protozoaninfections. For example, Winter et al., U.S. Pat. No. 5,977,077, whichis incorporated herein by reference, describes certain xanthone analogswhich have sub-10 μM IC₅₀s (some having sub-1μM IC₅₀s), againstPlasmodium and Leishmania. Despite these new xanthone analogs useful fortreating infectious diseases, particularly protozoan diseases, therestill is a need for new agents with comparable or better activities andreduced undesirable attributes, such as toxicity. A diverse array oftherapeutic agents also is desireable to prevent or reduce thedevelopment of drug-resistant protozoan strains.

SUMMARY

[0007] The present invention concerns new compounds which are useful,amongst other things, as antiparasitic agents. Methods for using thesenew compounds and certain known compounds as anti-parasitic agents aredescribed. These antiparasitic agents form complexes with heme and withporphyrins with superior affinity and are therefore useful in a varietyof other applications. The invention also is directed to compounds withbroad-spectrum anti-microbial activity.

[0008] As a result of studies aimed at developing new anti-parasiticagents, the present inventors have discovered that xanthones and a widerange of xanthone derivatives and structurally related compounds, asrepresented by Formula X below, have potent anti-parasitic activity. Thecompounds have broad-spectrum anti-microbial activity, includinganti-fungal activity against Candida albicans and Aspergillus fumigatus,and may possess antiviral activity as well.

[0009] Formula X

[0010] With reference to Formula X, A is oxygen, substituted antimony(stibium), sulfur or N-R' where R′ is H, OH, alkyl, haloalkyl, aryl orhaloaryl. Examples of substituted antimony groups include antimonialoxides and antimony substituted with hydroxy, chlorine, alkyl and arylgroups (e.g. SbCl, SbCl₃, SbOH, Sb(O)(OH)). R₁-R₈ are independentlyselected from the group consisting of H, OH, halogen, aryl, arylamine,alkyl, alkene, substituted alkyl (such as alkylamine, alkylthio,haloalkyl, and substituted alkyls having two or more of suchsubstituents), alkoxy, particularly lower alkoxy, such as methoxy,substituted alkoxy (such as alkoxylamine, cycloaminoalkoxy,dialkylaminoalkoxy, such as diethylaminoethoxy, haloalkoxy, and alkoxygroups having two or more of such substituents) amino, ester, ether,nitro groups and O-linked and C-linked carbohydrates. Alkyl and alkoxylgroups often include 10 or fewer carbon atoms in a straight or branchedchain, and are referred to as “lower” alkyl or alkoxy groups. Y isselected from the group consisting of NO, NOH, C═O, CH-OH, S═O, and SO₂.

[0011] Compounds having Y=carbonyl further satisfy Formula X1, where Aand R₁-R₈ are as stated above with reference to Formula X.

[0012] Formula X1

[0013] Certain Formula X1 compounds are compounds which also satisfyFormula X2:

[0014] Formula X2:

[0015] With reference to Formula X2, A is oxygen or sulfur, and R₁-R₆are independently selected from the group consisting of H, OH, halogen,aryl, arylamine, alkyl, alkene, substituted alkyl (such as alkylamine,alkylthio, haloalkyl, and substituted alkyls having two or more of suchsubstituents), alkoxy, substituted alkoxy (such as alkoxyamine,alkoxythio, haloalkoxy, and alkoxy groups having two or more of suchsubstituents) amino, ester, ether, nitro groups and O-linked andC-linked carbohydrates. Particular compounds satisfying Formula X2 haveR₁ and R₆ selected from the group consisting of H, —OH, OR, whre Rtypically is lower alkyl, such as OCH₃, and halogen. R₂-R₅ preferablyare selected from the group consisting of side chains linked to thearomatic rings by a carbonyl or thiocarbonyl (i.e., C═O or C═S,respectively), a methylene group (i.e., CH₂), oxygen, nitrogen orsulfur, and which further have a positively charged group on theterminal end of the linker. Such side chains are represented by FormulaX3.

[0016] Formula X3:

[0017] With reference to Formula X3, “A” is carbon, generally amethylene group, a carbonyl, amido, O, S, or N; “n” ranges from 1 to 10,preferably 2 to 8, and even more preferably from about 3 to 7 bothbranched chains or linear chains; and “P” is a group positively chargedat physiological pH, such as amines, amidines, guanidines,cycloalkylamines, such as dicyclopropyl amine, or cycloalkylimines, suchas pyrrolidine. “n” is selected to provide a chain length so that “P”preferably can interact with negatively charged groups, such as thepropionate groups of heme. Compounds having superior biological activityagainst Plasmodium and Leishmania have been made using alkoxy amines,such as those represented by Formula X4.

[0018] Formula X4:

[0019] With reference to Formula X4, “n” ranges from about 1 to 10,preferably 2 to 8, and even more preferably from about 3 to 7, includingboth branched chains and linear chains, and “R” typically is selectedfrom the group of hydrogen and alkyl or cyclo-alkyl groups, preferablylower alkyl groups, such as ethyl groups. Moreover, the R groups ofFormula X4 typically, but not necessarily, are the same or linked in acyclic structure, such as pyrrolidine.

[0020] Specific examples of such compounds include3,6-bis-N,N-diethylaminoxanthone,3,6-bis-β-(N,N-diethylamino)ethoxyxanthone,3,6-bis-γ-(N,N-diethylamino)propoxyxanthone,3,6-bis-δ-(N,N-diethylamino)butoxyxanthone,3,6-bis-ε-(N,N-diethylamino)amyloxyxanthone,3,6-bis-ζ-(N,N-diethylamino)hexyloxyxanthone,3,6-bis-η-(N,N-diethylamino)heptyloxyxanthone,3,6-bis-θ-(N,N-diethylamino)octyloxyxanthone, 3,6-bis-l-(N,N-diethylamino)nonyloxyxanthone, and3,6-bis-κ-(N,N-diethylamino)decyloxyxanthone. 4,5 bis substituted aminesand alkoxyamine analogs of these 3,6 bis-substituted compounds also arepreferred compounds, including 4,5-his-N,N-diethylaminoxanthone,4,5-bis-β-(N,N-diethylamino)ethoxyxanthone,4,5-bis-γ-(N,N-diethylamino)propoxyxanthone,4,5-his-δ-(N,N-diethylamino)butoxyxanthone,4,5-bis-ε-(N,N-diethylamino)amyloxyxanthone,4,5-bis-ζ-(N,N-diethylamino)hexyloxyxanthone,4,5-bis-η-(N,N-diethylamino)heptyloxyxanthone,4,5-bis-θ-(N,N-diethylamino)octyloxyxanthone,4,5-bis-l-(N,N-diethylamino)nonyloxyxanthone, and4,5-bis-κ-(N,N-diethylamino)decyloxyxanthone.

[0021] The disclosed invention also is directed to compositionscomprising the compounds described above, i.e., compositions includingcompounds according to Formulae X, X1 and X2. These compositions areuseful for the treatment of microbial diseases, such as malaria. Thesecompositions may include materials conventionally used to maketherapeutic compositions, and further may include additionaltherapeutics, particularly those useful for treating parasiticinfections, such as malaria and leishmania. Examples, withoutlimitation, of such therapeutics include chloroquine, antifolates,mefloquine, primaquine, cinchona alkaloids, such as quinine,sulfonamides, sulfones, tetracyclines, melarsoprol, nifurtimox,aminoacridines, aminoquinolines, sulfanolimides, pentamidine,stibogluconate, suramin, protease inhibitors, and mixtures thereof.

[0022] Also included in the present invention is a method of inhibitingthe growth of a microbial pathogen. The method comprises providing asufficient amount of a compound having Formulae X, X1 and/or X2, orcomposition comprising such compounds, and contacting the microbialpathogen with such compound(s) or composition(s). The present method isuseful for inhibiting microbial growth in vivo and in vitro. In oneaspect, the present invention provides a method for treating a patienthaving a microbial infection. “Patient” includes, without limitation,humans and animals, particularly economically important animals, such aslivestock and avians, particularly poultry infected with protozoans,such as Eimeria. The method comprises administering to the patient atherapeutically effective amount of a compound or compounds, orcomposition comprising such compound or compounds, satisfying FormulaeX, X1 and/or X2.

[0023] Another aspect of the present invention is the discovery thatcertain compounds having the xanthone ring structure depicted inFormulae X, X1 and/or X2 bind to, and inhibit the aggregation of, heme.A number of pathogens, including Plasmodium, a causative agent ofmalaria, degrade hemoglobin to obtain amino acids, and in so doingliberate toxic heme (Olliaro and Goldberg, 1995). To avoid the toxiceffects of the liberated heme, these pathogens have evolved a mechanismfor “aggregation” of heme units to form hemozoin. (Pagola, Stephens, P.W., Bohle, D. S., Kosar, A. D., Madsen, S. K., Nature, “The Structure ofMalaria Pigment Beta Haematin,” 404:307-310 (2000). The compoundsdisclosed herein which are shown to inhibit heme aggregation may thus beused to block heme aggregation and therefore to treat infections causedby these pathogens. These heme complexing compounds may kill pathogensby preventing these organisms from gaining access to the host's supplyof heme iron, or by causing a build-up of toxic levels of heme in theorganism. The compounds also may bind to heme of other metalloporphyrinsand block one-electron transfer reactions.

[0024] Compounds which are disclosed herein to inhibit heme aggregationmay be represented by the structure

X—Y—Z

[0025] where X is a group capable of interacting with the iron atom inheme (e.g., carbonyl, N→O, N—OH, SO₂, and S═O); Y is a substantiallyplanar aromatic system capable of interacting with the porphyrin ring ofheme, possibly through overlapping pi-pi orbitals; and Z represents oneor more groups capable of interacting with at least one carboxylate sidegroup of heme. In preferred embodiments, these compounds are Formula X2compounds.

[0026] The present invention also is directed to compositions useful fortreating diseases, such as malaria, which are caused by pathogens thatpolymerize heme. The compositions include a compound according toFormula X2. Also included in the present invention is a method forinhibiting the growth of such a pathogen comprising providing asufficient amount of a Formula X2 compound and contacting the pathogenwith this compound. Such a method is applicable to inhibit pathogengrowth in vivo and in vitro. In one aspect, the present inventionprovides a method for treating a patient having malaria, the methodcomprising administering to the patient a therapeutically effectiveamount of a compound according to Formula X2.

[0027] The invention also contemplates that Formula X, X1 and X2compounds can be administered to patients in a pro-drug form. Oneexample of a class of such prodrugs is correspondingly substitutedbenzophenones. These substituted benzophenones may react underphysiological conditions to produce active compounds (i.e., thecorresponding xanthone derivatives) satisfying Formulae X, X1 and/or X2(Winter et al., 1996).

[0028] Related to the ability of Formula X, X1 and X2 compounds to bindto heme is the ability of these compounds to bind to a porphyrin. Thisporphyrin binding activity may be exploited in the development oftreatments for porphyria. In addition, the binding between the FormulaX, X1 and/or X2 compounds and heme/porphyrins may find applications inother contexts, such as laundry detergents (e.g., to enhance the abilityof detergents to remove blood or grass stains) or in agriculturalproducts that bind chlorophyll (a metalloporphyrin) and perturb plantgrowth.

BRIEF DESCRIPTION OF THE FIGURES

[0029]FIG. 1 illustrates a proposed mechanism for the formation of2,3,4,5,6-pentahydroxyxanthone from the metabolic activation of exifoneby rufigallol within a red blood cell infected with the Plasmodiumparasite.

[0030]FIG. 2 is a schematic depiction of hemoglobin digestion (with theconcomitant release of heme) by the intracellular parasite Plasmodiumfalciparum.

[0031]FIG. 3 is a graph showing the inhibition of in vitro hemeaggregation by compound X5 (A═O) (2,3,4,5,6-pentahydroxyxanthone). Hemeand X5 (A═O) concentrations were 25 μM. Open diamonds indicate hemealone (control), filled diamonds represent heme and X5 (A═O) together.

[0032]FIG. 4 is a computer simulation of compound X6 (A═O)(2,3,4,5,6,7-hexahydroxyxanthone) complexing with free heme.

[0033]FIG. 5. provides the structure of 45-DEAE-X[4,5-bis-(β-diethylamino-ethoxy)-xanthone] and formation of thediprotonated form upon entry of this compound into the parasitedigestive vacuole.

[0034]FIG. 6 is a graph of chain length versus IC₅₀ for P. falciparum.

[0035]FIG. 7 is a graph of number of carbons in compound side chains for3,6-bis-diethylaminoalkoxyxanthones versus IC₅₀ values againstamastigote-infected peritoneal macrophages.

DETAILED DESCRIPTION

[0036] I. Definitions

[0037] The phrases “a compound according to Formula X” and “a xanthonederivative according to Formula X” refer to a compound having thefollowing structure:

[0038] Formula X:

[0039] With reference to Formula X, A is oxygen, substituted antimony(stibium), sulfur or N-R′ where R′ is H, OH, alkyl, haloalkyl, aryl orhaloaryl. Examples of substituted antimony groups include antimonialoxides and antimony substituted with hydroxy, chlorine, alkyl and arylgroups (e.g. SbCl, SbCl₃, SbOH, Sb(O)(OH)). R₁-R₈ are independentlyselected from the group consisting of H, OH, halogen, aryl, arylamine,alkyl, alkene, substituted alkyl (such as alkylamine, alkylthio,haloalkyl, and substituted alkyls having two or more of suchsubstituents), alkoxy, particularly lower alkoxy, such as methoxy,substituted alkoxy (such as alkoxylamine, cyclio aminoalkoxy,dialkylaminoalkoxy, such as diethylaminoethoxy, haloalkoxy, and alkoxygroups having two or more of such substituents) amino, ester, ether,nitro groups and O-linked and C-linked carbohydrates. Alkyl and alkoxylgroups often include 10 or fewer carbon atoms in a straight or branchedchain, and are referred to as “lower” alkyl or alkoxy groups. Y isselected from the group consisting of NO, NOH, C═O, CH-OH, S═O, and SO₂.

[0040] References to compounds such as “an X compound” refer to thecompounds shown in the Summary of the Invention section above. Where aparticular substituent in the formula is intended, it is givenparenthetically. For example X (A═O) refers to a compound according toFormula X where the A substituent is oxygen.

[0041] Compounds of the present invention which may be used to inhibitheme polymerization (and therefore to treat certain parasitic diseasessuch as malaria) are referred to as “Formula X compounds”. A Formula Xcompound is a compound broadly defined as:

X—Y—Z

[0042] where X is a group capable of interacting with the iron atom inheme; Y is a substantially planar aromatic system capable of interactingwith the porphyrin ring of heme through overlapping pi-pi orbitals; andZ represents one or more groups capable of interacting with at least onecarboxylate side group of heme, such as amine functionalities that areprotonated at physiological pH.

[0043] In preferred embodiments, Formula XH compounds also have thefollowing X1 structure, where Y=carbonyl (i.e., C═O), and A and R₁-R₈are as stated above with reference to Formula X.

[0044] More specifically, R₁-R₂ and R₇-R₈ are independently selectedfrom the group consisting of H, OH, and halogen, most typciallyhydrogen. At least one member (but preferrably both) of the R₃/R₆ orR4/R5 substituent pairs (and preferably both) is selected from the groupconsisting of amino, substituted amino, alkylamino, substituted alkylamino, arylamino, amidinium, alkylamidinium, guanidinium,alkylguanidinium, hydroxy, alkylhydroxy, alkoxyhydroxy, alkoxyamine,alkoxy-substituted amine, azido, carboxylic esters of hydroxy,alkylhydroxy and alkoxyhydroxy groups, COOH, alkyl-COOH, CONH₂ andalkyl-CONH2. The other member of the R₃/R₆ or R4/R5 substituent pairs isselected from the group consisting of H, OH, halogen, aryl, arylamine,alkyl, substituted alkyl (such as alkoxy, alkylamine, alkylthio andhaloakyl), amino, substituted amino, ester and nitro groups, O-linkedand C-linked carbohydrates, alkylamino, substituted alkyl amino,arylamino, amidinium, alkylamidinium, guanidinium, alkylguanidinium,alkylhydroxy, alkoxyhydroxy, alkoxyamine, alkoxy-substituted amine,azido, carboxylic esters of hydroxy, alkylhydroxy and alkoxyhydroxygroups, COOH, alkyl-COOH, CONH₂ and alkyl-CONH₂.

[0045] As used herein, the term “alkyl” encompasses alkanes, alkenes andalkynes, including linear and branched forms, isomers and stereoisomers.In certain embodiments, an alkyl is a lower alkyl, meaning an alkylhaving 10 or fewer carbon atoms.

[0046] The terms “ester” and “esterification” are used herein asordinarily understood in the chemical arts, see, for example, Morrisonand Boyd, Organic Chemistry, Allyn & Bacon, Inc., Boston, 1983, hereinincorporated by reference. Thus, an ester may be formed by, for example,the combination of an alcohol and an organic acid, with the concurrentelimination of water. The process of forming an ester is termed“esterification.” For example, the formula X1 compound2,3,4,5,6-pentahydroxyxanthone may be esterified by reaction withappropriate acid anhydrides resulting in the net replacement of one ormore hydroxyl substituents with ester substituents including, but notlimited to: acetoxy (OCOCH₃); propionyloxy (OCOCH₂CH₃); and butyryloxyOCO(CH₂)₂CH₃) substituents. Esters produced in this manner may begenerally represented by the formula OCO(CH₂)_(n)CH₃ wherein n is zeroor a positive integer. In particular embodiments, the term “ester” asused herein refers to an ester wherein n is 1-10.

[0047] A “microbial pathogen” is a microorganism capable of causingdisease in an animal. The term “microbial pathogen” includes bacterial,mycoplasmal, fungal, viral, helminth and protozoan organisms.“Parasites” are a subclass of microbial pathogens, being protozoan andhelminth organisms that are capable of invading, colonizing and, underappropriate conditions, causing disease in an animal. Examples ofparasites include, without limitation, Leishmania donovani, Leishmaniamexicana, Plasmodium falciparum, Giardia lamblia, Schistosoma mansonic,Trypanosoma gambiense and Trypanasoma cruzi. See generally, Robbins etal, Pathologic Basis of Disease (Saunders, 1984) 273-75, 360-83.

[0048] A “microbial infection” is a disease caused by a microbialpathogen.

[0049] A compound having “anti-microbial activity” is a compound that iscapable of inhibiting the growth of a microbial pathogen as determinedin in vivo or in vitro assays of the kind normally employed to determineminimum inhibitory concentrations (MICs) or 50% inhibitoryconcentrations (IC₅₀) of an antimicrobial agent.

[0050] An “oxidant agent” is an agent having the ability to produce orliberate free radical oxygen species or to render parasites or theirhost cells more susceptible to oxygen radical attack, or having thecapacity of oxidizing another compound. Examples of oxidant agents inthis general sense include ascorbic acid, hydrogen peroxide, primaquine(or its metabolites) and gamma radiation.

[0051] II. Biological Methods

[0052] A. Methods for Determining Biological Activity

[0053] The anti-parasitic activity of the compounds of the presentinvention was determined using three different parasites: Plasmodiumfalciparum, a causative agent of malaria; and Leishmania donovani andLeishmania mexicana, causative agents of leishmaniasis. The activity ofthe compounds against yeast was determined using Candida albicans.

[0054] 1. Assay for Anti-Malarial Activity

[0055] The D6 strain of P. falciparum was cultured in Group A⁺ humanerythrocytes and suspended at a 3.3% hematocrit in RPMI-1640 (Gibco,Grand Island, N.Y.) (containing 4 g/L glucose, 50 mg/L gentamicin and10% group A⁺human serum), buffered with 25 mM HEPES and 25 mM NaHCO₃(Trager and Jensen, 1976). Cultures were maintained at 37° C. in a gasmixture of 5% oxygen, 5% CO₂, and 90% nitrogen.

[0056] The in vitro anti-malarial activities of2,3,4,5,6-pentahydroxy-xanthone and other Formula X1 were measured bythe [³H]-ethanolamine incorporation method as described in Elabbadi etal., 1992, with minor modifications. [³H]-ethanolamine was obtained fromAmerican Radiolabeled Chemicals, Inc., St. Louis, Mo. Experiments wereconducted in 96 well plates in a total volume of 200 μl at a final redblood cell concentration of 2% (v/v). An initial parasitemia of 0.2 to0.5% was attained by addition of normal uninfected red cells.Radiolabeled ethanolamine was added after 48 hours of incubation and theexperiment was terminated after 72 hours by collecting the cells ontoglass fiber filters with an automated multiwell harvester.

[0057] Stock solutions of the various Formula X1 compounds weredissolved in DMSO at a concentration of 1 mM and diluted in completemedium (including 10% human serum) to provide 10X stock concentrationsin the range of 1 to 10,000 nM. The concentration of the formula X1compound giving 50% inhibition of label incorporation (IC₅₀) relative tocontrol (i.e., drug-free) conditions was calculated from thedose-response curve.

[0058] 2. Assay for Anti-Leishmania Activity

[0059] a. Promastigote Stage

[0060]Leishmania donovani was cultivated in Schneider's medium (Gibco,Grand Island, N.Y.) according to the methods described by Grogl et al.(1992). The in vitro susceptibility of L. donovani to Formula X1compounds was determined using the radiolabeled thymidine uptake assayessentially as described by Grogl et al. (1992). Briefly, promastigoteswere cultivated at 25° C. in Schneider's medium supplemented with 20%inactivated fetal calf serum and 100 μg/mL of gentamicin. Cells weremaintained in log phase by seeding at 1×10⁶/mL with subculturing whencultured densities approached 4×10⁷/mL before reaching their maximaldensity. For the assay, early log phase promastigotes were counted on ahemacytometer and resuspended at a concentration of 1-2×10⁶ cell/mL inassay media (Schneider's medium plus 10% fetal bovine serum). Ten-foldserial dilutions of each test compound were prepared as described aboveand added to 180 μL of the parasite suspension. After incubation for 24hours at 25° C., methyl-³H-thymidine was added to each sample for afinal concentration of 1-2 μCi per well. Each sample was then incubatedfor an additional 18 hours prior to harvesting. After this incubationtime, each sample was aspirated onto a filter mat, washed thoroughlywith deionized water, dried and then counted in a scintillation counterwith scintillation cocktail.

[0061] b. Intracellular Amastigote Stage

[0062] Bone-marrow-derived macrophages (BALB/c) were cultured withL-cell-conditioned medium for 5-7 days at 35° C. After this period ofincubation, mature macrophages were transferred to Labtek 4-chamberslides containing DMEM (Dulbecco's Modified Eagle's Medium) and 10%fetal calf serum. 50,000 cells were seeded in each chamber and allowedto attach overnight at 37° C. in a humidified incubator flushed with 5%CO₂. On the next day the macrophages were infected at an MOI(Multiplicity of Infection) of 10:1 with stationary promastigotes of L.mexicana. After twenty-four hours, the medium was removed and freshmedium was added containing various drug concentrations (0-5 μM).Following incubation for an additional forty-eight hours, one set ofslides were fixed in methanol, dried, and stained with Giemsa stain.Fresh medium and drug were added to a parallel set of chamber slides andincubated for another 48 hours before staining. Therefore, IC₅₀ valueswere obtained for each drug at 48 hours and 96 hours of drug exposure.Microscopic inspection permitted determination of % of infectedmacrophages and the number of amastigotes per 100 macrophages. The IC₅₀value is that concentration of drug that reduces the amastigote numberby 50% relative to drug-free controls. Results also were obtained inparallel fashion with macrophages.

[0063] 3. Assay for Anti-Candida Activity

[0064] The minimum inhibitory concentration (MIC) of formula X1compounds against a clinical isolate of Candida albicans was determinedusing the following method. As used herein MIC represents theconcentration of formula X1 compound that completely inhibits growth ofCandida albicans over the course of a 15-18 hour incubation period. Thedetermination of this concentration is made by visual inspection; thereis no visible growth in a tube containing the MIC of the formula X1compound whereas visible growth is present in tubes containing sub-MICconcentrations of the compound.

[0065]Candida albicans was grown to midlog-phase in Luria-Bertani broth(10 grams Bacto-tryptone, 5 grams Bacto-yeast extract and 10 grams NaClper liter) and then inoculated into sterile test tubes containing LBbroth to an initial density of 10³/ml. The formula X1 compound to betested is dissolved in dimethylsulfoxide (DMSO) at a concentration of 1mM and added to each tube at serial dilutions (1 μM, 10 μM, 25 μM, 50μM, 100 μM and 0 μM). The tubes are incubated at 35° C. for 15-18 hoursand then visually inspected.

[0066] B. Affinity of Drugs for Heme

[0067] 1. In Vitro Heme Aggregation Assay

[0068] Heme polymerization was carried out in 0.02 M phosphate buffer,pH 5.2 at 37° C. in the absence of protenis. A 10 mM stock solution ofhemin chloride in 0.1 M NaOH wa prepared freshly and incuated at 37° C.for at least 1 hour to effect complete dissolution. Xanthones weredissolved in dimethylformamide at 10 mM and diluted into 10 ml ofpre-warmed phosphate solution to a final concentraion of 25 μm.Polymerization wa sinitated gy addition of 25 μl of the hemin stocksolution to the test sample to yuiled a final concentratin of 25 μMheme. 25 μl of dimethylformaide wa added to the control smample. After7, 30, 60, 120 and 190 minutes of incugation at 37° C., a 1 ml aliquotwas withdraqwn, tranferred into an Eppendorf tube, and centrifugted at14,000 g for 2 minutes at room temperature to pellet the precipitate.The soluble fraction was then transferred to a semi-microcuvette(polymethyacrylate, VER), and its absoprtion was measured at 360 nmagainst a blank of the test compound in buffer. Control experimentsindiated that (1) the pH of the phosphate solution did not change uponaddition of the reagents or during thepolymerization process, and (ii)the amount of dimethylformamide used in this assay did not significantlyaffect the rate of polymerization. To estimate the effect of testcompounds on heme polymerization at a given time of incubation, thepercentage of soluble hemin remaining in thesample was calculated usingthe following formula: % sol.hemin=[A_((drug+hemin)t)-A_((drug)t)]/[A_((hemin)t=0)]×100% whereA_((drug+hemin)t) is the absorption (360 nm) of the soluble fraction inthe drug-hemin sample after various times of incubation; A_((drug)t) isthe absorption of the drug alone; and A_((hemin)t=0) is the absorptionof the hemin control sample (25 μM) measured immediately upon additionof the hemin stock solution.

[0069] The dose-dependent inhibition of heme polymerization wasevaluated as described above except the concentration of each drug wasvaried in the range of 0 to 1 mM. The reactions were allowed to proceedfor 2 hours in a 37° C. waterbath. After incubation, the polymer waspelleted as described above and the absorption (360 nm) of each solublefraction was measured against a blank containing the drug alone inbuffer. The IC₅₀ values were determined by nonlinear regression analysisof the dose-response curves of percent inhibition of heme polymerizationvs. drug concentration.

[0070] III Production of 2,3,4,5,6-pentahydroxyxanthone in ParasitizedErythrocytes Treated with RAufigallol and Exifone

[0071] As disclosed in Winter et al. (1996), rufigallol(1,2,3,5,6,7,-hexahydroxy-9, 10-anthraquinone) is a potentanti-parasitic agent and, when rufigallol is combined with exifone(2,3,3′,4,4′,5′-hexahydroxybenzophenone), a synergistic effect isobserved. The synergy between rufigallol and exifone is noted to produceabout a 350-fold increase in potency against malaria Plasmodiumparasites.

[0072] One aspect of the present invention is the discovery thatrufigallol and exifone interact in the parasitized erythrocyte to yield2,3,4,5,6-pentahydroxyxanthone, and that this compound is a potentanti-malarial agent. FIG. 1 shows a possible mechanism by which2,3,4,5,6-pentahydroxyxanthone could be produced when rufigallol andexifone are present in a parasitized erythrocyte. Basically, rufigallolis proposed to enter the parasitized erythrocyte, leading to theformation of hydrogen peroxide in a manner similar to thewell-documented redox cycling behavior of hydroxynaphthoquinones. In thepresence of catalytic quantities of adventitious iron or iron chelates,such as heme, (liberated as a result of the Plasmodium parasitedigesting hemoglobin, Atamna and Ginsburg, 1993), hydrogen peroxide isreadily decomposed to hydroxyl radicals (Goldstein et al., 1993; Aust etal., 1985). These highly reactive radicals are proposed to attackexifone and transform the diphenyl compound into2,3,4,5,6-pentahydroxyxanthone.

[0073] As reported in U.S. application Ser. No. 08/520,694, theanti-malarial activity of exifone can be potentiated by a very widerange of oxidant agents, including ascorbic acid, artemisinin anddoxorubicin. This observation is consistent with the mechanism proposedabove. The production of 2,3,4,5,6-pentahydroxyxanthone in the proposedreaction scheme was confirmed by incubating exifone with ascorbic acidin the presence of iron salt and oxygen in a buffered solution at 37-40°C. (the “Udenfriend system,” Brodie et al., 1954; Maisant et al., 1983;Udenfriend et al., 1954). Samples were removed from the reaction atvarious time points, lyophilized and extracted with acetone. Thesolubilized products were then methylated by addition of excesspotassium carbonate and dimethylsulfate in acetone and analyzed by gaschromatography-mass spectrometry. A peak corresponding to the methoxyderivative of 2,3,4,5,6-pentahydroxyxanthone was detected.

[0074] IV. Synthesis and Anti-Microbial Activity of2,3,4,5,6-pentahydroxyxanthone

[0075] 2,3,4,5,6-pentahydroxyxanthone was produced using the followingmethod.

[0076] A mixture of 1,2,3-Trimethoxybenzene (1.48 g) and2-hydroxy-3,4,5-trimethoxybenzoic acid (2.00 g) is stirred in 40 ml of≈9% solution of P₂O₅ in methanesulfonic acid at room temperature in astoppered flask for 4 hours. The 2-hydroxy-3,4,5-trimethoxybenzoic acidwas obtained by the method of Mayer and Fikentscher (Mayer andFikentscher (1956) Chem. Ber. 89:511) from 3,4,5-trimethoxybenzoic acidby bromination and then copper-catalyzed replacement of bromine (by OH)of 2-bromo-3,4,5-trimethoxybenzoic acid. The resultant orange mixture ispoured onto crushed ice (500 ml) producing an unfilterable gummyprecipitate. This crude product is then subjected to base-catalyzed ringclosure by heating in a beaker in 100 ml of 40% ethanol and 10 ml of 10NNaOH just below boiling point. As the mixture reaches 80° C., a whiteflocculent product appears. The temperature is maintained just below theboiling point and the volume is kept constant by addition of water.After 5 hours, the supernatant is bright yellow and a mass of theprecipitate has formed. Heating is continued for 4 more hours. Cooling,filtering (by suction) and washing with water afforded 1.37 g ofanalytically pure 2,3,4,5,6-pentainethoxyxanthone (yield approximately45% relative to benzoic acid). This base-catalyzed ring closure isillustrated below:

[0077] 2,3,4,5,6-pentahydroxyxanthone is then obtained by borontribromide treatment (200 ml of a 0.8 M solution in CH₂Cl₂) as thepentamethyl ether (0.45 g) is stirred at room temperature for 24 hours.After this period, the solution is poured into 100 ml of water andstirred for approximately 45 minutes before the precipitate is collectedby centrifugation. The supernatant is then decanted, the precipitate isshaken with water and centrifuged again. The final product is obtainedby freeze-drying of the wet precipitate to produce an orange powder(0.290 g, 81%).

[0078] The anti-malarial activity of 2,3,4,5,6-pentahydroxyxanthone wasdetermined by the method described above. The IC₅₀ was determined to be0.4-0.5 μM. Chloroquine, a standard anti-malarial agent has an IC₅₀ inthis assay system of approximately 0.04 μM.

[0079] The anti-leishmanial activity of 2,3,4,5,6-pentahydroxyxanthonewas determined by the intracellular amastigote method described above.The IC₅₀ was determined to be approximately 5 μM or 0.001 mg/ml.Mangostin, a naturally occurring xanthone, exhibited an IC₅₀ of 1 μM (or0.00041 mg/ml) in this same system. Grögl et al. (1992) report that twocommonly used anti-leishmanial drugs, Pentostam and Glucantine, haveIC₅₀ values in the range of approximately 0.1-2 mg/ml.

[0080] The MIC of 2,3,4,5,6-pentahydroxyxanthone against Candidaalbicans, determined using the method described above was found to beapproximately 37.5 μM. This corresponds to an IC₅₀ of approximately 10μg/ml.

[0081] V. Synthesis and Anti-Microbial Activityof2,3,4,5,6-pentaacetoxyxanthone

[0082] Although 2,3,4,5,6-pentahydroxyxanthone was found to have potentanti-malarial activity, the highly acidic nature of the 3 and 6 hydroxygroups of this compound (i.e. the R₃ and R₆ positions) could lead thesegroups to be highly ionized at physiological pH values. Such ionizationlikely would reduce the rate at which the compound could crossbiological membranes, thereby lowering the uptake of the compound intoparasitized erythrocytes. Accordingly, two derivatives of2,3,4,5,6-pentahydroxyxanthone were produced which were expected to bemore stable and uncharged above neutral pH: a pentacetoxy (i.e.esterified) derivative, 2,3,4,5,6-pentaacetoxyxanthone, as well as amethoxy (i.e. methyl ether) derivative, 2,3,4,5,6-pentamethoxyxanthone.The activity of these two derivatives against P. falciparum wasmeasured.

[0083] As shown in Table 1, the addition of the ether (methoxy) groupsessentially eliminated the anti-malarial activity of the compound,resulting in an IC₅₀ of >100 μM. This reduction in activity is believedto be attributable to the extreme stability of the methoxy groups; themethoxy group is less amenable to enzymatic cleavage under physiologicalconditions.

[0084] The pentaacetoxy derivative was produced by heating2,3,4,5,6,-pentahydroxyxanthone in acetic anhydride in the presence of acatalytic amount of sulfuric acid, followed by recrystallization. Incontrast to the methoxy derivative, the esterified2,3,4,5,6,-pentaacetoxyxanthone was several times more potent than2,3,4,5,6-pentahydroxyxanthone (exhibiting an IC₅₀ of approximately0.075 μM). The enhanced activity of the esterified compound ispostulated to be due to the ability of the compound to cross membranes(due to its neutral charge at physiological pH). Esters are also knownto be amenable to enzymatic cleavage under physiological conditions.Accordingly, it is expected that the pentaacetoxyxanthone enters thecell where it is enzymatically cleaved to produce pentahydroxyxanthone.

[0085] VI Synthesis and Anti-Microbial Activityof2,3,4,5,6,7,-hexaahydroxyxanthone

[0086] The newly discovered anti-malarial activity of2,3,4,5,6-pentahydroxyxanthone prompted the investigation of otherxanthones and related compounds. One such related compound was2,3,4,5,6,7,-hexahydroxyxanthone which was prepared using the followingmethod.

[0087] 2-hydroxy-3,4,5-trimethoxybenzoic acid (1.14 g, 0.005 mol) and1,2,3,4-tetramethoxybenzene (0.99 g, 0.005 mol) and 25 ml of a 9%solution of P₂O₅ in methanesulfonic acid were shaken in a 50 mlcylindrical glass tube with a Teflon-lined screw-cap at room temperaturefor 54 hours. The dark orange mixture was then poured onto crushed ice(150 ml). After melting, the product was extracted with methylenechloride (3×40 ml). After removal of the solvent, the residue waschromatographed on silica gel (30 g) with CH₂Cl₂. Of the three fractionsobtained (the eluent was monitored by thin-layer chromatography), themiddle one was uniform and left pure 2-hydroxy-3,4,5,2′,3′,4′,5′-heptamethoxybenzophenone (0.51 g, 25%) as a yellow oil uponevaporation of the solvent. This was dissolved in 100 ml 75% alcoholwhereafter 5 ml of 10N NaOH were added and the mixture was heated toboiling in a beaker for three hours; the volume was kept at 100 ml byoccasional addition of water. The mixture was then transferred to a 250ml round bottom flask and refluxed for another 17 hours. After cooling,suction filtration yielded 0.36 g of 2,3,4,5,6,7-hexamethoxyxanthone asa white product (small needles, matted, 77%). In a deprotectionprocedure, similar to the one described above for pentamethoxyxanthone,0.42 g of the hexamethoxyxanthone produced 0.296 g ofhexahydroxyxanthone (91%) as a pale yellow powder. It was foundadvantageous to circumvent the need for centrifugation by stirring themethylene chloride-water mixture (from the quenching of theBBr₃-solution) in a wide-mouthed container for several hours, leading tothe evaporation of the methylene chloride; the mixture is then easilyfilterable.

[0088] The antimalarial activity of 2,3,4,5,6,7-hexahydroxyxanthone wasdetermined by the method described above. The IC₅₀ was determined to be0.075 μM. The IC₅₀ of this compound against Leishmania was determined tobe approximately 5 μM. The MIC of the compound against Candida albicanswas determined to be approximately 37.5 μM, corresponding to an IC₅₀ ofapproximately 10 μg/ml.

[0089] VII Scope of Formula X Compounds

[0090] The inventors have discovered that a wide range of compoundsrelated to 2,3,4,5,6-pentahydroxyxanthone have anti-microbial activity.These compounds can be represented by Formula X

[0091] With reference to Formula X, A is oxygen, substituted antimony(stibium), sulfur or N-R′ where R′ is H, OH, alkyl, haloalkyl, aryl orhaloaryl. Examples of substituted antimony groups include antimonialoxides and antimony substituted with hydroxy, chlorine, alkyl and arylgroups (e.g. SbCl, SbCl₃, SbOH, Sb(O)(OH)). R₁-R₈ are independentlyselected from the group consisting of H, OH, halogen, aryl, arylamine,alkyl, alkene, substituted alkyl (such as alkylamine, alkylthio,haloalkyl, and substituted alkyls having two or more of suchsubstituents), alkoxy, particularly lower alkoxy, such as methoxy,substituted alkoxy (such as alkoxylamine, cycloaminoalkoxy,dialkylaminoalkoxy, such as diethylaminoethoxy, haloalkoxy, and alkoxygroups having two or more of such substituents) amino, ester, ether,nitro groups and O-linked and C-linked carbohydrates. Alkyl and alkoxylgroups often include 10 or fewer carbon atoms in a straight or branchedchain, and are referred to as “lower” alkyl or alkoxy groups. Y isselected from the group consisting of NO, NOH, C═O, CH-OH, S═O, and SO₂.

[0092] The activities of various Formula X compounds against thePlasmodium falciparum parasite are shown in Table 1 (reproduced below)of U.S. Pat. No. 5,977,077, which is incorporated herein by reference.The activities of various Formula X compounds against Leishmaniadonovani are shown in Table 2 of U.S. Pat. No. 5,977,077. Forcomparison, Table 2 also shows the activity of stibogluconate, astandard anti-leishmanial. The activities of various Formula X compoundsagainst Plasmodium falciparum and intracellular are provided in Table 3.The activities of various Formula X compounds against Plasmodiumfalciparum and intracellular Leishmania mexicana parasites correlated totheir affinity for heme (hematin) are provided in Table 3.

[0093] A. Rational Design Improvements and Enhanced Activity of Formulax Compounds

[0094] Based on a molecular model that was constructed depicting thecarbonyl-iron coordination, the π-π stacking of coplanar aromaticmolecules, and hydrogen bonding between the hydroxyl substituents of4,5-dihydroxyxanthone and hematin's propionate groups, we proceeded withsynthesis of nitrogenated xanthones. 3-6-bis-diethylaminoxanthone(referred to as “C0” in Table 4) was prepare but it exhibited only weakantimalarial activity (IC₅₀≈20 μM) against P. falciparum. These findingsindicated that the protonatable species could not be directly attachedto the aromatic ring or in conjugation with the carbonyl moiety.Presumably this arrangement results in formation of bis-cations (formedonce the drug enters the acidic environment of the parasite digestivevacuole) which draw electrons away from the carbonyl and diminish theinteraction with heme iron.

[0095] A decision was made to retain the oxygen atom (a conjugativelyelectron-donating species) as a bridge for an R-group containing theprotonatable nitrogen. Molecular models constructed from a ball andstick kit reproducing bond lengths, geometries, and angle strainsdemonstrated that an optimal chain length in the range of C4 to C6 wouldpermit a relatively strain-free, close association between the ammoniumions of the xanthone and the carboxylate groups of heme. For comparativepurposes, the straight-chain di-substituted series from C₂ to C₈ (i.e.,excepting C₇), each with a terminal diethylamino group was prepared.Shown in Table 4 is the remarkable relationship between chain length andantimalarial activity within the series . . . and the extraordinarypotency of the C5 (3,6-bis-ω-diethylaminoamyloxyxanthone) and C6(3,6-bis-ω-diethylaminohexyloxyxanthone) congeners. FIG. 6 displays thecorrelation graphically with minimum IC₅₀ values of ≈0.1 μM recorded forthe pair against the mefloquine resistant D6 clone of P. falciparum. Thedegree of potency was found to be the same against the multidrugresistant W2 strain.

[0096] B. Antileishmanial Activity of Modified Xanthones AgainstLeishmania Parasites in the Amastigote Stage of Development

[0097] Intracellular amastigote studies. Leishmania parasites exhibit adimorphic life cycle, existing as promastigotes in the insect vector andas amastigotes in the phagolysosome of the host macrophage. Because theamastigote stage is the only relevant clinical form of the parasites, weexamined the activity of the nitrogenated xanthones against theheme-auxotrophic, intracellular amastigote form of L. mexicana.Stationary promastigotes of L. mexicana M379 rapidly invaded murineperitoneal macrophages obtained from BALB/c mice and incubated at 35° C.producing heavily burdened cells with 10 to 50 amastigotes permacrophage within 24 hours. An experiment was performed to investigatethe ability of selected xanthones to reduce the number of intracellularamastigotes using this model. The IC₅₀ value is that concentration ofdrug required to reduce the intracellular parasite burden by 50%relative to a drug-free control chamber. We tested the xanthone seriesagainst amastigote-infected murine peritoneal macrophages. Macrophageswere seeded into Labtek chamber slides (25,000 per well/8 chamber slide)and allowed to incubate overnight in Dulbecco's Modified Eagle Medium(DMEM) supplemental with 10% fetal calf serum. On the next day the cellswere infected with L. mexicana promastigotes at an MOI of 5:1 and thenincubated for 24 hours at 35° C. After incubation, the monolayers werewashed with buffer and medium containing varying concentrations of eachxanthone (0 to 10 μM) was added to each chamber. After 48 hours ofincubation (i.e., Day 3 of the experiment) one set of slides wasremoved, fixed with methanol, and stained with Giemsa. For another setof slides, we replenished the culture medium, added fresh drug to eachwell as appropriate, and then incubated the chambers for another 48hours before staining with Giemsa. IC₅₀ values were then averaged for 3separate experiments and the results are presented in Table 4 andgraphically in FIG. 7. The relative potency for each of the compoundsobserved on the 5^(th) day of the experiment was quite similar to thevalue obtained for 48 hours of drug exposure in the peritonealmacrophage system. The IC₅₀ values recorded for C5 and C6 exposure for96 hours of treatment were 6 nM and 2 nM.

[0098] The strong correlation between hematin affinity andantileishmanial potency for the xanthone series is strong evidence thatheme salvage is perturbed by the most active compounds. Furthermore, theremarkable potency of C5 and C6 (both diacidic weak bases) againstintracellular amastigotes leads us to speculate that the drugs areacting by accumulating in the phagolysosome-as prescribed in theoriginal design of this series. These nitrogenated xanthones havediethylaminoalkoxy groups extending from the 3 and 6 positions of thepharmacophore. So positioned, under mildly acidic conditions of pH suchas those found in the digestive vacuole of the Plasmodium parasites orthe phagolysosome of Leishmania infected macrophages, the positivelycharged ammonium cations align themselves in opposition to thepropionate side chains of heme in a net-neutral electrostaticinteraction yielding an especially stable heme: drug complex. Note thatthe charge nature of the drug that results on entry into acidic regionsof the cell or parasite causes the drug to accumulate. It has notescaped our attention that the target of our drugs is immutable andthat, in the case of the leishmania, the xanthones may not need to enterthe parasite to cause its death (the drug merely needs to accumulate inthe phagolysosome).

[0099] Other examples of specific Formula X compounds are illustratedbelow:

TABLE 1 IC₅₀, μM vs. Compound Name Xanthone Structure PlasmodiumXanthone

>10 Mangostin

5 Mangiferin

50 3,4,5,6,-Tetrahdroxy-xanthone

10 2,3,4,5,6-Pentahydroxy-xanthone

0.4 to 0.5 2,3,4,5,6,7-hexahydroxy-xanthone

0.075 2,3,4,5,6-Pentamethoxy-xanthone

>100 2,3,4,5,6-Penta-acetoxyxanthone

0.075 1,2,3,5,6,7-Hexahydroxy-xanthone

25-50 1,3-dihydroxyxanthone

>100 1,3,5,6,7-pentahydroxy-xanthone

1

[0100] TABLE 2 IC₅₀ mg/ml for Chemical Name Structure Leishmania2,3,4,5-penta-hydroxyxanthone “X %”

0.0015 2,3,4,5,6,7-hexa-hydroxyxanthone “X6”

0.0015 Mangostin

0.00041 Mangiferin

>0.05 Stibogluconate [Sodium Antimony (V) Gluconate]

0.1 to 1.0 literature reported value

[0101] TABLE 3 IC₅₀ μM, IC₅₀, μM P. falciparum in vitro heme Compoundname Compound structure clone D6 polymerization 2-hydroxyxanthone

50 >1000 3-hydroxyxanthone

>100 >1000 1,3-dihydroxyxanthone

75 >1000 3,6-dihydroxyxanthone

>100 >500 4,5-dihydroxyxanthone

16 14 2,3,4-trihydroxyxanthone

40 17 3,4,5,6-tetrahydroxyxanthone

5 2.5 2,3,4,5,6-pentahydroxyxanthone (X5)

0.4 1.2 1,3,5,6,7-pentahydroxyxanthone

1 9 2,3,4,5,6,7-hexahydroxyxanthone (X6)

0.1 1.4 2,3,4,5,6-pentamethoxyxanthone

>100 >1000 2,3,4,5,6-pentaacetylxanthone

0.075 >1000

[0102] Table 4 provides antiparasitic activities for selectednitrogenated xanthones versus intraerythrocy Plasmodium falciparum(strain D6) and intracellular amastigotes of Leishmania mexicana M379.The xanthones illustrated in Table 4 typically included substitutedalkyls, such as alkyl amines and alkoxyamines. TABLE 4 IC₅₀ μM* IC₅₀μM** P. falciparum L. Chemical Name Structure D6 mexicana3,6-bis-N,N-diethylamino-xanthone (36-DEAX)

20 μM not tested 3,6-bis-β-(N,N-diethylamino)ethoxy-xanthone (36-DEAE-X)

2.2 μM ˜1.25 μM 3,6-bis-γ-(N,N-diethylamino)propoxy-xanthone (36-DEAP-X)

1.5 μM 0.5 μM 3,6-bis-δ-(N,N-diethylamino)butoxy-xanthone (36-DEAB-X)

0.65 μM 0.2 μM 3,6-bis-ε-(N,N-diethylamino)-amyloxy-xanthone(36-DEAAmyl-X) C5

0.10 μM 0.06 μM 3,6-bis-(N,N-diethylamino)-hexyloxy-xanthone(36-DEAHexyl-X) C6

0.075 μM 0.02 μM 3,6-bis-(N,N-diethylamino)octyloxy-xanthone(36-DEAOctyloxy-X) C8

0.43 μM Not tested 4,5-bis-β-(N,N-diethylamino)ethoxy-xanthone(45-DEAE-X)

2.8 μM not tested 4,5-bis-β-(N,N-diethylamino)amyloxy-xanthone

575 nM not tested 3,5-bis-(N,N-diethylamino)amyloxy-xanthone

825 nM Not tested 3-(N,N-diethylamino)amyloxy-xanthone

2.5 μM Not tested

[0103] VIII. Sources of Formula X1 Compounds and Preferred Method ofSynthesis

[0104] Many xanthones and xanthone derivatives can be purchasedcommercially from sources including: ICN Biomedicals, Irvine, Calif.,U.S.A.; Sigma Chemical Company, St. Louis, Mo., U.S.A.; Aldrich ChemicalCompany, Milwaukee, Wis., U.S.A.; and Janssen Chimica (Belgium). Inaddition, many xanthones are naturally occurring compounds which can bepurified by methods such as those described in Hostettmann et al.(1995).

[0105] A. General Xanthone Synthesis Method

[0106] Xanthones according to the present invention may be synthesizedby the general method described above, for the synthesis of2,3,4,5,6,7-hexahydroxyxanthone and 2,3,4,5,6-pentahydroxyxanthone.Essentially, this method comprises subjecting ano-hydroxy-o′-methoxy-benzophenone to base treatment (e.g., aqueoussodium hydroxide), which leads to the formation of the centraloxygen-bridged ring; the o-phenoxide (from the o-hydroxyl in basicmedium) then replaces the methoxide on the other ring by nucleophilicsubstitution. The net effect is expulsion of CH₃O—, and the formation ofa diphenyl ether. Since the two phenyl rings are already linked by acarbonyl group, a xanthone is obtained. The o—OH,o′—OCH₃ groupings arerequired for this reaction; although the methyl could be replaced withother groups, this is not likely to be of any advantage since methylethers are readily available. However, other substituents can be presentin the two aromatic rings of the benzophenones. For example, for thesynthesis of the penta- and hexa-hydroxyxanthones described above, theseother substituents were methoxy groups.

[0107] The benzophenones used in synthesizing the xanthones as describedabove may be obtained by combining substituted benzoic acids andmethoxybenzenes by a condensation or other coupling procedure. In anexemplary condensation procedure, the benzoic acid carries an o-hydroxygroup:

[0108] This coupling can be achieved by condensation in polyphosphoricacid or a mixture of phosphorus pentoxide and methanesulfonic acid.Alternatively, benzophenones may be synthesized by Friedel-Craftsacylation (of a benzoyl chloride and a polymethoxybenzene), or by theHoesch synthesis, or by reaction of a benzoyl chloride with anappropriately metalated (e.g., lithiated) aromatic, or other methods.

[0109] In particular cases, additional substituents may be introducedinto the benzophenone after the benzophenone has been synthesized.

[0110] Alternatively, xanthones may be derived from benzophenones byoxidative cyclization. This method essentially requires an o-hydroxygroup on one ring and a free position (occupied by H) on the other ring.Oxidation (e.g., with K₃[Fe(CN)₆], or KMnO₄) produces an oxygen bridgewith the elimination of 2H.

[0111] B. Synthesis of Thioxanthones

[0112] Thioxanthones may be obtained by a number of methods. Exemplarysyntheses include: (1) combining an o-mercaptobenzoic acid with ahalobenzene (preferably iodo or bromo); and (2) combining ano-halobenzoic acid (preferably either bromo or iodo) with amercaptobenzene. The intermediate diphenylsulfide produced in each caseis then condensed to yield the required thioxanthone as illustrated inthe following schematic:

[0113] Methods of synthesizing thioxanthones using this general reactionscheme are described in Hollis-Showalter et al., J. Med. Chem., 31, 1527(1988).

[0114] C. Synthesis of Acridones

[0115] Acridones may be synthesized by a number of different methods.The following methods are exemplary and are well known in the art.

[0116] Acridones may be formed from o-nitrobenzophenones, which arereduced to obtain o-aminobenzophenones which are in turn cyclized witheither o′-methoxy or o′-hydroxy groups to produce the acridones. Theo-nitrobenzophenones which are used as starting materials may beobtained either by Friedel-Crafts acylation of phenols ormethoxybenzenes using o-nitrobenzoylchlorides, or by direct nitration ofbenzophenones, or by coupling of lithiated arenes with o-nitrobenzolchlorides (e.g., as described by Parkham et al., Journal of OrganicChemistry 46, 1057 (1981). An exemplary synthesis is illustrated below:

[0117] Alternatively, o-nitrobenzophenones may be formed by coupling of2-methyl-3, 1-benzoxazin-4-ones (from o-aminobenzoic acid by heatingwith acetic anhydride) with aromatic Grignard reagents (e.g., Adams etal. J.C.S. Perkin Trans I 2089 (1976)).

[0118] Alternatively, acridones may be produced by zinc chloridecatalyzed condensation of hydroxyanthranilic acids andpolyhydroxybenzenes (such as described by Bahar et al., Phytochemistry21, 2729 (1982)) and illustrated in the following scheme:

[0119] Acridones may also be formed by cycloaddition of derivatives ofanthranilic acids with dehydrobenzenes such as described by Khanapure etal., Tetrahedron Letters 31:2869 (1990).

[0120] D. Deprotection

[0121] Deprotection of polymethoxyxanthones, polymethoxythioxanthones orpolymethoxyacridones may be achieved in a number of ways, includingtreating with either hydriodic acid or with a methylene chloridesolution of boron tribromide, and hydrolysis of the intermediateboron-phenoxy compound.

[0122] E. Synthesis of Alkoxyamine-Substituted Xanthones

[0123] A number of methods can be used to make alkoxyamine substitutedxanthones. The first method comprises reacting a correspondingdihydroxyxanthone with an alkyl amine having a suitable leaving group,such as a halogen, in the presence of a base and typically with theaddition of heat. Working embodiments of this general method have usedchlorinated alkyl amines. These chlorinated alkyl amines were reactedwith the corresponding hydroxyxanthones in an alcoholic solution in thepresence of sodium hydroxide.

[0124] The second general method is a two step process. The first stepinvolves reacting a corresponding hydroxyxanthone with an excess of analkyl group having two leaving groups attached thereto, such as twohalogens, e.g., 1,3-dibromoethane. This first step generally isconducted in acetonic solvent with a carbonate base, such as potassiumcarbonate. The resulting halogenated ethers are then reacted with anappropriate amine, such as diethyl amine, to displace the second halogenleaving group and form the alkoxyamine-substituted xanthones. The secondstep has been done in an ether solvent, such as tetrahydrofuran. Heatcan be used to increase reaction rate and/or yield of both steps.

[0125] F. Replacing Carbonyl Oxygen with Sulfur to Form Thiocarbonyls

[0126] Formula X indicates that the Y group can be either oxygen orsulfur, i.e., thiocarbonyls. Thiocarbonyl can be made from thecorresponding ketones using a number of reagents, including withoutlimitation, Lawesson's reagent [see, Lawesson et al. Bull. Soc. Chim.Belges 87:223 (1978); Lawesson et al. Bull. Soc. Chim. Belges 87:229(1978); Lawesson et al. Bull. Soc. Chim. Belges 87:293 (1978)],2,4-bis(4-methoxyphenyl)-1,2,3,4-dithiadiphosphonate-2, 4-disulfide,bis(tricyclophexyltin)-sulfide [(R₃Sn)₂S, where R=cycolhexyl] or P₄S₁₀.

[0127] IX Activity of Formula X Compounds

[0128] The Formula X compounds according to the present invention areuseful in inhibiting the growth of microbial pathogens, includingprotozoan parasites (for example, Plasmodium sp. and Leishmania sp.) andyeast (for example, Candida albicans ). Thus, one aspect of the presentinvention is a method of inhibiting the growth of a microbial pathogenby contacting the microbial pathogen with a Formula X compound. In thiscontext, it is, of course, necessary to contact the microbial pathogenwith a sufficient amount of the Formula X compound to inhibit growth ofthe pathogen. One skilled in the art will readily appreciate that theamount of compound sufficient to inhibit the growth of the microbialpathogen will vary according to the Formula X compounds selected, thetarget microbial pathogen and the environment in which the microbialpathogen is growing. Standard methods are available for determining theIC₅₀ concentration of Formula X1 compounds for microbial pathogens invitro. Alternatively, ED₅₀ values may be determined in an animal. SeeMunson, Principles of Pharmacology (Chapman and Hall, 1995) Chapter 1.Exemplary IC₅₀ values (showing activities against Plasmodium andLeishmania, respectively) are presented in Tables 1 and 2 of U.S. Pat.No. 5,977,077, and Table 3 above These values relate to the inhibitionof a microbial pathogen grown in vitro. Contacting the microbialpathogen with a compound according to Formula X1 may also be performedin vivo where necessary to inhibit the growth of microbial pathogensunder physiological conditions. The “Pharmaceutical Compositions”section below addresses compositions and dosages appropriate forinhibiting the growth of microbial pathogens in such circumstances.

[0129] X. Heme Polymerization and Formula X Compounds

[0130] Certain parasites, including Plasmodium spp. and Schistosomaspp., obtain amino acids for growth by degrading hemoglobin from the redblood cells of the infected host. In the case of the malarial parasite,degradation of hemoglobin takes place in the parasite's digestivevacuole, which is an acidic proteolytic compartment essential to themetabolism of the parasite (see FIG. 2). As the hemoglobin is brokendown, free toxic heme is released. To prevent the build-up of toxicheme, the parasites polymerize the heme for storage in a non-toxic formcalled hemozoin.

[0131] While the Formula X compounds of the present invention exhibitanti-microbial activity against a range of pathogens, it has now beendiscovered that a certain sub-group of these compounds form complexeswith free heme, which results in the inhibition of heme aggregation. Inturn, this leads to the accumulation of toxic heme in the parasite'sdigestive vacuole and ultimately to the death of the parasite. Thesecompounds include those listed in Table 3 belong to a group related toFormula X compounds which are referred to herein as “Formula XHcompounds”. Formula XE compounds may be particularly effective againstthose parasites, including Plasmodium and Schistosoma, which rely onhemoglobin catabolism to survive in the infected host or must rely onthe host's heme iron reserves for synthesis of critical ferroproteins.

[0132] Table 3 below shows the ability of a range of formula X1compounds to inhibit heme aggregation, as determined using the simple invitro heme aggregation assay described above. Under the conditions ofthis assay, heme aggregation was found to be pH dependent(polymerization required a pH of between 4.5 and 5.5). Aggregationoccurred spontaneously and was more than 95% complete within 2 hours ofcommencement of incubation with the compound X5 (A═O)(see FIG. 3).

[0133] The IC₅₀ values shown in Table 3 are the average of at least twoindependent determinations of full dose-response curves. Xanthone andthe tested monohydroxyxanthones did not exhibit any inhibitory activityin this assay. Moderate inhibitory activity (i.e., IC₅₀ 8-20 μM) wasobserved for the compounds bearing a single hydroxy group at either 4-or5-position, whereas the greatest activity was observed for xanthonescontaining hydroxy groups at both positions. For example,2,3,4-trihydroxyxanthone exhibited an IC₅₀ of 16.5 μM, while2,3,4,5,6-pentahydroxyxanthone (X5, A═O) yielded a value of 1.2 μM.Consistent with this structure-activity profile, the 4,5-hydroxylatedxanthones also exhibited the most pronounced in vitro antimalarialactivity. Furthermore, pentamethoxy-X5 and pentaacetyl-X5 were inactivein this assay, though the latter was shown to be a potent antimalarialagent. Presumably, pentaacetyl-X5 is hydrolysable in infected red bloodcells by a non-specific esterase, whereas pentamethoxy-X5 is not.

[0134] These findings suggest that X5 (A═O) and other compounds shown inTable 3 form soluble complexes with heme monomers or oligomers andinterfere with hemozoin formation. Such action may result in the deathof the parasite by one of several mechanisms, including preventingdetoxification of free heme, starving the parasite for iron, orincreasing the osmotic pressure within the parasite digestive vacuole.The relative abilities of these compounds to inhibit in vitro hemepolymerization are in good correlation with their in vitro antimalarialactivities, and are indicative of the following structure-activityrelationships: (I) in general, a higher degree of hydroxylation isfavored for the inhibitory activity; and (ii) hydroxylation in the lowerhalf of the pharmacophore may be central to full activity. Based onthese observations, a model for the interaction of these compounds ispresented in FIG. 4. This model shows the interaction of X6 (A═O) withheme and serves to illustrate the following interactions: (1) betweenthe heme iron and the carbonyl oxygen; (2) between the two planararomatic systems; and (3) between the carboxylate side groups of theheme and the 4-and 5-position hydroxyls of the xanthone. Theseinteractions may take any form of known chemical interaction, includingcovalent bonding, hydrogen bonding, ionic bonding, and polar andnonpolar bonding. Moreover, this model predicts that chemicalmodifications at the 3, 4, 5 or 6 positions which improve associationwith the heme carboxylate groups will result in even greaterantimalarial activity.

[0135] Accordingly, Formula XH compounds can be defined as compoundswhich inhibit heme polymerization and which have the following structure

X—Y—Z

[0136] where X is a group capable of interacting with the iron atom inheme; Y is a substantially planar aromatic system capable of interactingwith the porphyrin ring of heme, possibly through overlapping pi-piorbitals; and Z represents one or more groups capable of interactingwith at least one carboxylate side group of heme.

[0137] In preferred embodiments, a Formula XH compound has the followingstructure:

[0138] where A is oxygen, substituted antimony (stibium), sulfur or N-R′wherein R′ is H, OH, alkyl, haloalkyl, preferably lower alkyl or lowerhaloalkyl wherein “lower” means 10 or fewer carbon atoms, aryl orhaloaryl; R₁—R₈ are independently selected from the group consisting ofH, OH, halogen, aryl, arylamine, alkyl, substituted alkyl (such asalkylamine, alkylthio and haloakyl, and alkyl groups having two or moresuch substituents), alkoxy, substituted alkoxy (such as alkoxyamine,alkoxythio and haloalkoxy, and alkoxy groups having two or more suchsubstituents) amino, ester and nitro groups and O-linked and C-linkedcarbohydrates. Preferably, at least one member of the R₃/R₆ or R₄/R₅substituent pairs, and even more preferably both members of the pair, isselected from the group consisting of amino, substituted amino,alkylamino, substituted alkyl amino, arylamino, amidinium,alkylamidinium, guanidinium, alkylguanidinium, hydroxy, alkylhydroxy,alkoxyhydroxy, alkoxyamine, cycloalkoxyamine, alkoxy-substituted amine,azido, carboxylic esters of hydroxy, alkylhydroxy and alkoxyhydroxygroups, COOH, alkyl—COOH, CONH₂ and alkyl—CONH₂. The other member of theR₃/R₆ and R₄/R₅ substituent pair is selected from the group consistingof H, OH, halogen, aryl, arylamine, alkyl, substituted alkyl (such asalkoxy, alkylamine, alkylthio and haloakyl), amino, substituted amino,ester and nitro groups, O-linked and C-linked carbohydrates, alkylamino,substituted alkyl amino, arylamino, amidinium, alkylamidinium,guanidinium, alkylguanidinium, alkylhydroxy, alkoxyhydroxy, alkoxyamine,alkoxy-substituted amine, azido, carboxylic esters of hydroxy,alkylhydroxy and alkoxyhydroxy groups, COOH, alkyl—COOH, CONH₂ andalkyl—CONH₂.

[0139] Examples of Formula XH compounds include, without limitation,4,5-dihydroxyxanthone, 2,3,4-trihydroxyxanthone,3,4,5,6-tetrahydroxyxanthone, 2,3,4,5,6-pentahydroxyxanthone,1,3,5,6,7-pentahydroxyxanthone, 2,3,4,5,6,7-hexahydroxyxanthone,3,6-bis-N,N-diethylaminoxanthone,3,6-bis-β-(N,N-diethylamino)ethoxyxanthone,3,6-his-γ-(N,N-diethylamino)propoxyxanthone,3,6-bis-δ-(N,N-diethylamino)butoxyxanthone,3,6-bis-ε-(N,N-diethylamino)amyloxyxanthone,3,6-bis-ζ-(N,N-diethylamino)hexyloxyxanthone,3,6-bis-η-(N,N-diethylamino)heptyloxyxanthone,3,6-bis-θ-(N,N-diethylamino)octyloxyxanthone,3,6-bis-l-(N,N-diethylamino)nonyloxyxanthone,3,6-bis-κ-(N,N-diethylamino)decyloxyxanthone, 4,5 bis substituted aminesand alkoxyamine analogs, such as 4,5-bis-N,N-diethylaminoxanthone,4,5-bis-β-(N,N-diethylamino)ethoxyxanthone,4,5-bis-γ-(N,N-diethylamino)propoxyxanthone,4,5-bis-δ-(N,N-diethylamino)butoxyxanthone,4,5-bis-ε-(N,N-diethylamino)amyloxyxanthone,4,5-bis-ζ-(N,N-diethylamino)hexyloxyxanthone,4,5-bis-η-(N,N-diethylamino)heptyloxyxanthone,4,5-bis-θ-(N,N-diethylamino)octyloxyxanthone,4,5-bis-l-(N,N-diethylamino)nonyloxyxanthone, and4,5-bis-κ-(N,N-diethylamino)decyloxyxanthone.

[0140] Methods of synthesizing these compounds are known in the art andare described in a representative manner above in relation to Formula Xcompounds. Additional detail concerning the synthesis of compoundssatisfying Formula X are provided below in the examples.

[0141] Based on empirical results and the mechanism proposed for theinteraction of Formula XH compounds with heme, the 3,6 and4,5-bis-substituted alkylamine and alkoxyatnine compounds appearparticularly effective in complexing with heme.4,5-bis-(N,N-diethylamino)ethoxyxanthone is a diprotic base, which uponentry into the acidic vacuole becomes positively charged, effectively“trapping” the drug within this compartment where it will complex withheme. The positively charged residues are arranged to be in oppositionto the heme carboxylate side chains so as to facilitate formation of asoluble heme:xanthone complex (the ionic nature of the trapped xanthonewill also maintain the drug:heme complex in solution). 4,5-DEAE-X may bereadily prepared from 4,5-dihydroxyxanthone (which as described above,is synthesized by base-catalyzed cyclization of the appropriateortho-hydroxy-methoxylated-benzophenone). To produce 4,5-DEAE-X,4,5-Di-hydroxyxanthone was reacted under basic conditions with ethylenedibromide to yield 4,5-bis-(β-bromoethoxy)-xanthone. The latter was thenreacted with diethylamine to yield the desired product.

[0142] The present invention thus encompasses the use of Formula XHcompounds to inhibit heme polymerization and to inhibit the growth ofthose pathogens which polymerize heme, such as Plasmodium. It also isapparent that the Formula XH compounds may be used to treat infectionscaused by pathogens which require access to the host heme iron reservesfor survival.

[0143] XI. Pharmaceutical Compositions

[0144] Formula X and XH compounds having anti-microbial activity areadministered to patients in conventional dosage forms prepared bycombining an appropriate dose of the compound with standardpharmaceutical carriers. Suitable pharmaceutical carriers may be, forexample, solids or liquids. Suitable solid carriers include lactose,magnesium stearate, terra alba, sucrose, talc, stearic acid, gelatin,agar, pectin, acacia and cocoa butter. The amount of solid carrier willvary widely depending on which carrier is selected, but preferably willbe from about 25 mg to about 1 gram. Suitable liquid carriers includesyrup, peanut oil, olive oil, sesame oil, propylene glycol, polyethyleneglycol and water. The carrier or diluent may also include time delaymaterial well known to the art such as, for example, glyceryl,monostearate or glycerol distearate, either alone or with a wax. Theforegoing examples of suitable pharmaceutical carriers are onlyexemplary and one of skill in the art will recognize that a very widerange of such carriers may be employed.

[0145] The formulation of the Formula X and XH compounds with apharmaceutical carrier can take many forms. For example, the formulationmay be a tablet, capsule, powder, suppository, lozenge, syrup, emulsion,liquid suspension or solution, or sterile injectable liquid. Thepharmaceutical compositions are prepared by conventional techniquesinvolving procedures such as mixing, granulating and compressing, anddissolving the ingredients. As will be appreciated from the foregoingexemplary formulations, administration of the compounds can be by anyknown route, including oral administration, intramuscular andintravascular injection.

[0146] The methods of treating a patient suffering from a microbialdisease, such as malaria, in accordance with this invention compriseadministering to the patient a therapeutically effective amount of acompound according to Formula X or Formula XH. Preferably, the patientwill be administered the compound in a formulation as described above(i.e. in combination with a pharmaceutical carrier), the formulationhaving a therapeutically effective amount of the compound. As usedherein, “a therapeutically effective amount” preferably is an amountthat results in complete remission of the disease. However, it will berecognized that any improvement in the patient's condition is clinicallyadvantageous. Hence, “a therapeutically effective amount” alsoencompasses amounts of the administered compound that result in partialremission of the disease or which slow or limit the further progressionof the disease, or which inhibit the growth of the infectious agent orwhich reduce the clinical signs and symptoms of the disease (forexample, fever and chills in a malaria infection). It is anticipatedthat therapeutically effective dosages which slow or limit the spread ofthe disease, or which inhibit the growth of the parasite will beparticularly suitable for combination with other anti-microbial drugs.

[0147] The compounds of the invention can be administered in a dailydosage schedule of from about 10 mg to about 10 g. One skilled in theart will recognize that in determining the active amount of theanti-microbial compound to be administered, the activity of the specificcompound selection, the age, weight and condition of the patient and theadministration of other drugs to the patient should be considered.

[0148] The Formula X and XH compounds also may be indirectly provided topatients in pro-drug formulations. For example, Formula X and XHxanthones may be produced by co-administration of an oxidant agent witha corresponding substituted benzophenone under physiological conditions.A pro-drug is thus defined herein as a compound which reacts underphysiological conditions to produce a Formula X or XH compound. Thus,the pro-drug for 4,5-DEAE-X would be3,3′-bis-(β-diethylamino)ethoxy-2-hydroxy-benzophenone, and the pro-drugfor 2,3,4,5,6-pentahydroxyxanthone would be2,3,3′,4,4′,5′-hexahydroxybenzophenone (exifone). The provision of the Xand XH compounds in pro-drug form (i.e. the corresponding benzophenones)may be particularly useful where the oxidant agent which is administeredwith the pro-drug is another anti-microbial agent. For example, thewidely used anti-malarial agent primaquine is such an oxidant agent, andthe combination of an XH pro-drug with primaquine is expected to be aparticularly efficacious treatment for malaria.

Examples

[0149] The following examples are provided to illustrate certainfeatures of the present invention. These examples should be consideredillustrative only. The scope of the present invention should bedetermined not by reference to the following examples, but rather byreference to the attached claims.

Example 1

[0150] This example describes the synthesis of3,6-bis-β-(N,N-diethylamino)ethoxy-xanthone by the first general methoddescribed above. 0.30 gram of 3,6-dihydroxyxanthone [See, R. Meyer etal., Ber., 30:989 (1897)] and 1.1 grams of NaOH were combined in analcoholic solvent and warmed. 2.0 grams of (C₂H₅)₂NCH₂CH₂Cl•HCl wereadded to the mixture, and the mixture was then heated to reflux forforty minutes. Thereafter, another 4 grams of NaOH and 8 grams of(C₂H₅)₂NCH₂CH₂Cl•HCl were added to the refluxing mixture. After standingovernight, the mixture was poured into water (about 400 milliliters) andextracted twice with methylene chloride (100 milliliters; 50milliliters). The combined methylene chloride extracts were extractedwith 250 milliliters of 8% HCl. The resulting aqueous phase was againextracted with methylene chloride (2×50 milliliters). The aqueous phasewas combined with 200 milliliters of 5N NaOH and extracted with ether(3×75 milliliters). The combined organic extracts were evaporated atroom temperature to provide 0.39 grams of substantially pure, colorlesscrystals of 3,6-his-β-(N,N-diethylamino)ethoxyxanthone (70%).

[0151] The crude product was purified using silica gel chromatography.The silica gel was wetted with methylene chloride and the sample placedon the column using methylene chloride. The column was then flushed with100 milliliters of hexanes. The compounds were eluted with triethylamineand hexanes (1:1). After evaporation, 0.23 gram of a product pure by TLCand proton NMR was obtained. The product obtained had an Rf of about0.4.

[0152]3,6-bis-γ-(N,N-diethylamino)propoxyxanthone,4,5-bis-β-(N,N-diethylamino)ethoxyxanthone, and4,5-bis-γ-(N,N-diethylamino)propoxyxanthone have been made in a mannersubstantially identical to that described in Example 1.γ-N,N-diethylamino-1-chloropropane was made according to the methoddescribed by A. Marxer, Helvetica Chimica Acta, 24:709 (1941).Substituted xanthones having more than three methylene units in thealkoxyamine side chain are difficult to make according to this firstmethod in view of competing side reactions that occur with thehalogenated amine reagent.

Example 2

[0153] This example described the synthesis of3,6-bis-ε(N,N-diethylamino)pentyloxy-xanthone. 0.30 gram of3,6-dihydroxyxanthone, 5.16 grams of 1,ω-dibromopentane, 50.0milliliters of acetone and 5.0 grams of potassium carbonate were heatedwith stirring for eight hours. After cooling, the mixture was vacuumfiltered and the solvent and excess dibromopentane were removed. Theresulting solid was recrystallized from aqueous acetone to provide 0.80gram of substantially pure product (about an 87% yield). This productwas then used without further purification in the next step.

[0154] 3,6-bis-(ω-bromopentoxy)xanthone, 5.0 milliliters of anhydroustetrahydrofuran and 11 milliliters of diethylamine were refulxed for 12hours. After cooling, precipitated material was filtered off and thefiltrate evaporated using a stream of air. The crude product was thenpurified using silica-gel chromatography. The silica was wetted withmethylene chloride, and the sample transferred to the column, usingmethylene chloride. The column was first flushed with 350 milliliters ofmethylene chloride, second with 110 milliliters of hexane to displacethe methylene chloride, and the compound was eluted with a 1:1triethylamine:hexane mixture.

[0155] The compound so obtained was then converted to the hydrochloridesalt. 0.34 gram of the compound was dissolved in 10 milliliters ofmethanol. The mixture was then placed in an ice bath, and gaseoushydrochloric acid was then introduced for seven minutes with stirring.The brightly yellow solution was set aside to air dry. After about 24hours, a yellow oil was obtained, which was placed under vacuum toprovide 0.399 gram of the hydrochloride salt.

[0156] The present invention has been described with reference tocertain preferred embodiments. It will be appreciated that the scope ofthe invention can be broader than that described. For example, certainalkoxydiethylamine xanthones are described. A person of ordinary skillin the art will realize that additional amines are within the scope ofthe present invention, such as alkoxydipropylamine xanthone,cycloalkylamines, such as dicyclopropyl amine, or cycloalkylimines, suchas pyrrolidine. The true scope of the present invention therefore shouldbe determined with reference to the following claims.

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1. A compound according to Formula X

where A is oxygen, substituted antimony (stibium), sulfur or N-R′, whereR′ is H, OH, alkyl, haloalkyl, aryl or haloaryl; R₁-R₈ are independentlyselected from the group consisting of H, OH, halogen, aryl, arylamine,alkyl, alkene, substituted alkyl, alkylthio, alkoxy, substituted alkoxy,cycloaminoalkoxy, substituted alkylthio, amino, amido, ester, ether andnitro groups and O-linked and C-linked carbohydrates; and Y is selectedfrom the group consisting of NO, NOH, C═O, CH-OH, S═O, and SO₂.
 2. Thecompound according to claim 1 where Y is S═O.
 3. The compound accordingto claim 1 where A is sulfur.
 4. The compound according to claim 1 whereY is carbonyl.
 5. The compound according to claim 1 where A is oxygen.6. The compound according to claim 1 where A is oxygen and Y iscarbonyl.
 7. The compound according to claim 6 where at least one ofR₃-R₆ is dialkylaminoalkoxy.
 8. The compound according to claim 6 wheretwo or more of R₃-R₆ are alkoxyamines.
 9. The compound according toclaim 6 selected from the group consisting of3,6-bis-N,N-diethylaminoxanthone,3,6-bis-β-(N,N-diethylamino)ethoxyxanthone,3,6-bis-γ-(N,N-diethylamino)propoxyxanthone,3,6-bis-δ-(N,N-diethylamino)butoxyxanthone,3,6-bis-ε-(N,N-diethylamino)amyloxyxanthone,3,6-bis-ζ-(N,N-diethylamino)hexyloxy-xanthone,3,6-bis-η-(N,N-diethylamino)heptyloxyxanthone,3,6-bis-θ-(N,N-diethylamino)octyloxyxanthone,3,6-bis-l-(N,N-diethylamino)nonyloxyxanthone,3,6-bis-κ-(N,N-diethylamino)decyloxyxanthone,4,5-bis-N,N-diethylaminoxanthone,4,5-bis-β-(N,N-diethylamino)ethoxyxanthone,4,5-bis-γ-(N,N-diethylamnino)propoxyxanthone,4,5-bis-δ-(N,N-diethylamino)butoxyxanthone,4,5-bis-ε-(N,N-diethylamino)amyloxyxanthone,4,5-bis-ζ-(-(N,N-diethylamino)hexyloxyxanthone,4,5-bis-η-(N,N-diethylamino)heptyloxy-xanthone,4,5-bis-θ-(N,N-diethylamino)octyloxyxanthone,4,5-bis-l-(N,N-diethylamino)nonyloxyxanthone, and4,5-bis-κ-(N,N-diethylamino)decyloxyxanthone.
 10. The compound accordingto claim 1 where the compound is 3,6-bis-N,N-diethylaminoxanthone.
 11. Acomposition comprising a pharmaceutical carrier and a compound havingFormula X

where A is oxygen, substituted antimony (stibium), sulfur or N-R′, whereR′ is H, OH, alkyl, haloalkyl, aryl or haloaryl; R₁-R₈ are independentlyselected from the group consisting of H, OH, halogen, aryl, arylamine,alkyl, alkene, substituted alkyl, alkylthio, alkoxy, substituted alkoxy,cycloaminoalkoxy, substituted alkylthio, amino, amido, ester, ether andnitro groups and O-linked and C-linked carbohydrates; and Y is selectedfrom the group consisting of NO, NOH, C═O, CH-OH, S═O, and SO₂.
 12. Thecomposition according to claim 11 having two or more compounds havingFormula X.
 13. The composition according to claim 11 further including aconventional antimicrobial agent.
 14. The compound according to claim 11where A is oxygen.
 15. The compound according to claim 11 where A isoxygen and Y is carbonyl.
 16. The compound according to claim 15 whereat least one of R₃-R₆ is an alkoxyamine.
 17. The compound according toclaim 15 where two or more of R₃-R₆ are alkoxyamines.
 18. The compoundaccording to claim 15 selected from the group consisting of3,6-bis-N,N-diethylaminoxanthone,3,6-bis-β-(N,N-diethylamino)ethoxyxanthone,3,6-bis-γ-(N,N-diethylamino)propoxyxanthone,3,6-bis-δ-(N,N-diethylamino)butoxyxanthone,3,6-bis-ε-(N,N-diethylamino)amyloxyxanthone,3,6-bis-ζ-(N,N-diethylamino)hexyloxy-xanthone,3,6-bis-η-(N,N-diethylamino)heptyloxyxanthone,3,6-bis-θ-(N,N-diethylamino)octyloxyxanthone,3,6-bis-l-(N,N-diethylamino)nonyloxyxanthone,3,6-bis-κ-(N,N-diethylamino)decyloxyxanthone,4,5-bis-N,N-diethylaminoxanthone,4,5-bis-β-(N,N-diethylamino)ethoxyxanthone,4,5-bis-γ-(N,N-diethylamino)propoxyxanthone,4,5-bis-δ-(N,N-diethylamino)butoxyxanthone,4,5-bis-ε-(N,N-diethylamino)amyloxyxanthone,4,5-bis-ζ-(N,N-diethylamino)hexyloxyxanthone,4,5-bis-η-(N,N-diethylamino)heptyloxy-xanthone,4,5-bis-θ-(N,N-diethylamino)octyloxyxanthone,4,5-bis-l-(N,N-diethylamino)nonyloxyxanthone, and4,5-bis-κ-(N,N-diethylamino)decyloxyxanthone.
 19. A method forinhibiting the growth of a microbial pathogen, comprising: providing acompound according to Formula X, or a composition comprising a compoundaccording to Formula X

where A is oxygen, substituted antimony (stibium), sulfur or N-R′, whereR′ is H, OH, alkyl, haloalkyl, aryl or haloaryl; R₁-R₈ are independentlyselected from the group consisting of H, OH, halogen, aryl, arylamine,alkyl, alkene, substituted alkyl, alkylthio, alkoxy, substituted alkoxy,cycloaminoalkoxy, substituted alkylthio, amino, amido, ester, ether andnitro groups and O-linked and C-linked carbohydrates; and Y is selectedfrom the group consisting of NO, NOH, C═O, CH-OH, S═O, and SO₂; andcontacting the pathogen with an effective amount of the compound orcomposition.
 20. The method according to 19 where A is oxygen.
 21. Thecompound according to claim 19 where A is oxygen and Y is carbonyl. 22.The compound according to claim 21 where at least one of R₃-R₆ is analkoxyamine.
 23. The compound according to claim 21 where two or more ofR₃-R₆ are alkoxyamines.
 24. The compound according to claim 21 selectedfrom the group consisting of 3,6-bis-N,N-diethylaminoxanthone,3,6-bis-β-(N,N-diethylamino)ethoxyxanthone,3,6-bis-γ-(N,N-diethylamino)propoxyxanthone,3,6-bis-δ-(N,N-diethylamino)butoxyxanthone, 3,6-bis-ε-(N,N-diethylamino)amyloxyxanthone,3,6-bis-ζ-(N,N-diethylamino)hexyloxy-xanthone,3,6-bis-η-(N,N-diethylamino)heptyloxyxanthone,3,6-bis-θ-(N,N-diethylamino)octyloxyxanthone,3,6-bis-l-(N,N-diethylamino)nonyloxyxanthone,3,6-biS-κ-(N,N-diethylamino)decyloxyxanthone,4,5-bis-N,N-diethylaminoxanthone,4,5-bis-β-(N,N-diethylamino)ethoxyxanthone,4,5-bis-γ-(N,N-diethylamino)propoxyxanthone,4,5-bis-δ-N,N-diethylamino)butoxyxanthone,4,5-bis-ε-(N,N-diethylamino)amyloxyxanthone,4,5-bis-ζ-(N,N-diethylamino)hexyloxyxanthone,4,5-bis-η-(N,N-diethylamino)heptyloxy-xanthone,4,5-bis-θ-(N,N-diethylamino)octyloxyxanthone,4,5-bis-l-(N,N-diethylamino)nonyloxyxanthone, and4,5-bis-κ-(N,N-diethylamino)decyloxyxanthone.
 25. A compound having astructure X—Y—Z where X is a group capable of interacting with the ironatom in heme; Y is a planar aromatic system capable of interacting withthe porphyrin ring; and Z is one or more groups capable of interactingwith at least one carboxylate side group of heme.
 26. The compoundaccording to claim 25 and further having Formula XI

where A is oxygen, substituted antimony (stibium), sulfur or N-R′, whereR′ is H, OH, alkyl, haloalkyl, aryl or haloaryl; and R₁-R₈ areindependently selected from the group consisting of H, OH, halogen,aryl, arylamine, alkyl, alkene, substituted alkyl, alkoxy, substitutedalkoxy, cycloaminoalkoxy, dialkylaminoalkoxy, substituted alkylthio,amino, ester, ether and nitro groups and O-linked and C-linkedcarbohydrates.
 27. The compound according to claim 26 where at least oneof R₃-R₆ is an alkoxyamine.
 28. The compound according to claim 26 wheretwo or more of R₃-R₆ are alkoxyamines.
 29. The compound according toclaim 26 selected from the group consisting of3,6-bis-N,N-diethylaminoxanthone,3,6-bis-β-(N,N-diethylamino)ethoxyxanthone,3,6-bis-γ-(N,N-diethylamino)propoxyxanthone,3,6-bis-δ-(N,N-diethylamino)butoxyxanthone,3,6-bis-ε-(N,N-diethylamino)amyloxyxanthone,3,6-bis-ζ-(N,N-diethylamino)hexyloxy-xanthone,3,6-bis-η-(N,N-diethylamino)heptyloxyxanthone,3,6-bis-θ-(N,N-diethylamino)octyloxyxanthone,3,6-bis-l-(N,N-diethylamino)nonyloxyxanthone, 3,6-bis-κ-(N,N-diethylamino)decyloxyxanthone,4,5-bis-N,N-diethylaminoxanthone,4,5-bis-β-(N,N-diethylamino)ethoxyxanthone,4,5-bis-γ-(N,N-diethylamino)propoxyxanthone,4,5-bis-δ-(N,N-diethylamino)butoxyxanthone,4,5-bis-ε-(N,N-diethylamino)amyloxyxanthone,4,5-bis-ζ-(N,N-diethylamino)hexyloxyxanthone,4,5-bis-η-(N,N-diethylatnino)heptyloxy-xanthone,4,5-bis-θ-(N,N-diethylamino)octyloxyxanthone,4,5-bis-i-(N,N-diethylamino)nonyloxyxanthone, and4,5-bis-κ-(N,N-diethylamino)decyloxyxanthone.
 30. A method for forming asoluble heme complex, comprising contacting heme with a compound havinga structure X—Y—Z where X is a group capable of interacting with theiron atom in heme; Y is a planar aromatic system capable of interactingwith the porphyrin ring; and Z is one or more groups capable ofinteracting with at least one carboxylate side group of heme.
 31. Amethod for forming a soluble porphyrin complex, comprising contactingporphyrin with a compound having a structure X—Y—Z where X is a groupcapable of interacting with the iron atom in heme; Y is a planararomatic system capable of interacting with the porphyrin ring; and Z isone or more groups capable of interacting with at least one carboxylateside group of heme.